When producing a pharmaceutical composition, it is not enough to formulate the drug substance into the drug product, it is also vital that the resulting drug product is approved by the regulatory body in the country in which the pharmaceutical composition is to be used. In the United States, the appropriate regulatory body is the United States Food and Drug Administration (FDA) (www.fda.gov/), and in Europe, it is for instance the European Agency for the Evaluation of Medicinal Products (EMEA) (www.emea.eu.int/).
The approval process is intensely regulated and the drug developers will be required to submit a substantial amount of information regarding the drug product to the regulatory authorities in order to obtain approval. This may include information regarding the potency of the drug product and assays to determine this potency.
Such a potency assay serves to characterize the product, to monitor lot-to-lot consistency and to assure stability of the product, and should therefore be sufficiently sensitive to detect differences which may impact mechanism of action and function of the product and are thereby of potential clinical importance. In addition, it is desirable that the potency assay bears the closest possible relationship to the putative physiological/pharmacological activity of the product.
A suitable potency assay should meet the following primary criteria:                ability to measure potency value within the product specifications.        high sensitivity for detection of differences of potential clinical importance.        close relationship with the mechanism of action and putative physiological/pharmacological activity of the product.        
A potency assay selected on basis of the primary criteria should also meet the following secondary criteria:                sufficiently low intra- and inter-assay variation (to obtain precision needed to support product specifications).        sufficient robustness        amendable to high-throughput analysis.        
The development of techniques to produce recombinant monoclonal antibodies has prompted a significant amount of research into the therapeutic use of monoclonal antibodies directed against disease targets. Several pharmaceutical compositions comprising monoclonal antibodies have subsequently been approved for marketing or are in clinical development all over the world. The therapeutic utility of an antibody as a drug depends on the ability of the antibody to bind the antigen, but often antibody Fc-mediated activities also play a critical role in the mechanism of action. Indeed, whereas the effect of some antibody drug products are achieved by simply binding of the antibody to the antigen resulting in for instance blocking the access of ligands to the antigen, the performance of certain antibody drug products may in addition depend on effector functions, such as for instance binding of Fc receptors and/or induction of complement activation.
Zanolimumab (also referred to as HuMax-CD4) is a fully human monoclonal antibody with an IgG1 heavy chain and a light chain of the kappa-type (IgG1,κ) directed against human CD4 (EP0854917). Zanolimumab is manufactured in a mammalian cell (CHO) culture and purified by affinity, ion exchange and size exclusion chromatography. The zanolimumab drug substance is formulated at 20 mg/ml in a phosphate buffered saline, pH 7.4 to become the zanolimumab drug product. One mechanism of zanolimumab is to deplete and/or inactivate CD4+ T cells. This may occur via for instance antibody dependent cell-mediated cytotoxicity (ADCC), down-modulation of CD4 expression on the T cell surface, and/or interference with CD4 signal transduction, T cell activation, and T cell proliferation. All these categories of mechanisms of action depend on binding of CD4 antigen by the antigen-binding moiety of zanolimumab located in the Fab fragment. In addition, ADCC and CD4-downregulation require binding of the Fc region of zanolimumab to an Fc receptor. The induction of ADCC has been identified as an important mechanism of action for Zanolimumab.
Zalutumumab (also referred to as HuMax-EGFr) is a human antibody directed against human EGFr with a heavy chain of the IgG1 isotype and a light chain of the kappa type (IgG1,κ) (WO02/100348). Zalutumumab is currently manufactured in a mammalian cell (CHO) suspension culture, expressing Zalutumumab using the GS vector system, and purified by affinity and ion exchange procedures, including specific viral inactivation and removal procedures. The drug product Zalutumumab (20 mg/mL) is formulated by diluting the Zalutumumab drug substance (25 mg/mL) in a buffer containing 50 mM sodium phosphate, 50 mM sodium chloride, 3% (w/v) mannitol, 0.02% (w/v) polysorbate 80 and 0.01% (w/v) EDTA and adjustment to pH 6.0.
Anti-tumor effect in mice was also observed at low HuMax-EGFr-receptor occupancy, which is likely based on the engagement of immune effector mechanisms, in particular ADCC. So, one mechanism of action of HuMax-EGFr, ADCC, is through Fc-FcR interactions.
The potency of antibodies for which Fc binding to Fc receptor plays a critical role for the mechanism of action are traditionally measured by use of biological assays in which the effect assessed is dependent on Fc-Fc receptor binding. Such assays may include ADCC, induction or inhibition of T cell activation requiring antibody cross-linking or the induction or inhibition of cytokine production such as production of interleukin 2 (IL-2). However, such assays are relatively cumbersome, are highly variable because of expected variations due to cell culture or because primary cells are often required. The latter in particular introduces variability because of variations in donor immune status, polymorphisms in expressed genes and variations in cell-type purity. Such biological assays therefore may be less optimal for batch release purposes. There is thus a need for fast, efficient and sensitive potency assays showing a close relationship to the mechanism of action and putative physiological/pharmacological activity of the antibody drug product for use in the production of pharmaceutical compositions particularly comprising antibodies in which mechanism of action is dependent on binding to Fc receptors.